Metal organic framework ratio detection fluorescent probe as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of fluorescent materials, and particularly relates to a metal organic framework ratio detection fluorescent probe as well as a preparation method and application thereof. The fluorescent sensing material is prepared from zirconium tetrachloride, 2, 5-dihydroxy terephthalic acid and 5, 10, 15, 20-tetra (4-carboxyl phenyl) porphyrin through a double-ligand reaction of zirconium tetrachloride, 2, 5-dihydroxy terephthalic acid and 5, 10, 15, 20-tetra (4-carboxyl phenyl) porphyrin. The material can realize the ratio detection of Cu < 2 + >, the concentration change of Cu < 2 + > in a solution to be detected can be obtained by determining the ratio of the fluorescence intensity of the solution at 648nm to the fluorescence intensity at 537nm under the excitation wavelength of 410nm, the material is successfully applied to cell fluorescence imaging, the selectivity and the anti-interference capability of the material are strong, the detection sensitivity and the accuracy are relatively high, and the application prospect is wide. And the detection limit of a target detection object is far lower than the normal concentration range of the copper ion content in the body of a WD patient.

Description

Technical Field

[0001] The invention belongs to the technical field of fluorescent materials, and in particular relates to a metal organic framework ratio detection fluorescent probe and a preparation method and application thereof. Background Art

[0002] Copper is an essential trace element for living organisms. However, once the concentration of copper ions exceeds the threshold maintained by the evolutionarily conserved homeostatic mechanism, it will become toxic. As an autosomal recessive genetic disease, patients with Wilson disease (WD) have disturbed copper metabolism, which results in large accumulation in organs such as the liver and brain. Because copper has redox activity, it can easily lead to liver damage and mental abnormalities in WD patients; especially in the liver, Cu 2+ Excessive accumulation of Cu can lead to various liver diseases, such as liver damage or inflammation, cirrhosis, fatty liver, etc. What's more, patients in the late stage of WD disease may die from cirrhosis and liver failure. 2+ Imaging tests are of great significance to human health.

[0003] At present, typical instrumental detection techniques such as atomic absorption spectroscopy (AAS), inductively coupled plasma mass spectrometry (ICP), atomic emission spectroscopy (AES), colorimetry, and fluorescence can be used to analyze Cu 2+ Among them, the fluorescence method has attracted much attention due to its simple operation and high sensitivity. Metal organic frameworks (MOFs) are new porous hybrid materials self-assembled from organic ligands and inorganic metal ions. In recent years, the research on MOFs materials has expanded to materials science, engineering, physics, biomedicine and other related disciplines.

[0004] At present, many MOF-based fluorescent probes for copper ions have been developed. However, there are few reports on their fluorescence imaging in vivo, and most of the reported probes are either single signal detection or have complex synthesis methods. It is of great significance to develop MOF-based copper ion fluorescent probes that are easy to synthesize, have good selectivity, and high sensitivity. Summary of the invention

[0005] The purpose of the present invention is to provide a metal organic framework ratio detection fluorescent probe and its preparation method and application, so as to overcome the shortcomings of the prior art. The prepared ratio fluorescent probe can realize the detection of Cu 2+ It can be used for quantitative detection and cell imaging, with high anti-interference, high selectivity and high detection sensitivity.

[0006] In order to achieve the above object, the technical solution of the present invention is:

[0007] In a first aspect, the present invention provides a method for preparing a metal organic framework ratio detection fluorescent probe, comprising the following steps:

[0008] ZrCl4 and 2,5-dihydroxyterephthalic acid are dissolved in an organic solvent, acetic acid and deionized water are added to dissolve, 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin is added to dissolve, and then heated to react to prepare a metal organic framework ratio detection fluorescent probe.

[0009] In some other embodiments, the molar ratio of ZrCl4, 2,5-dihydroxyterephthalic acid and 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin is 1:2:(0.016-0.16).

[0010] Or, the organic solvent is N,N-dimethylformamide;

[0011] Or, the amount of acetic acid used is 1-5mL;

[0012] Or, the amount of deionized water used is 0-0.5 mL.

[0013] In some other embodiments, the heating reaction temperature is 115-125° C., and the reaction time is 22-26 h.

[0014] In some other embodiments, after the heating reaction is completed, the obtained product is further purified, and the purification process includes centrifuging, washing, soaking, washing again and drying the obtained product;

[0015] The solvent used for washing or re-washing is N,N-dimethylformamide;

[0016] The immersion solvent is N,N-dimethylformamide, the amount of N,N-dimethylformamide is 20-50mL, and the immersion time is 20-28h;

[0017] The drying is performed at 50-70° C. in vacuum until constant weight is reached.

[0018] In some other embodiments, the preparation method of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin is: dissolving pyrrole in propionic acid, adding p-formylbenzoic acid, and reacting at 130-160° C. for 2-4 hours under a nitrogen atmosphere to obtain the obtained product;

[0019] Or, after the reaction is completed, the product may be washed and dried;

[0020] The solvent used for the washing is dichloromethane, and the drying is drying at 50-70° C. under vacuum overnight.

[0021] In a second aspect, the present invention provides a metal-organic framework ratio detection fluorescent probe prepared by the method for preparing the metal-organic framework ratio detection fluorescent probe described in the first aspect.

[0022] In a third aspect, the present invention provides the use of the metal organic framework ratio detection fluorescent probe described in the second aspect in the detection of copper ions in water or biological samples. When the detection sample is a biological sample, the purpose is not to diagnose and treat diseases.

[0023] In a fourth aspect, the present invention provides a method for detecting copper ions in water, comprising the following steps:

[0024] The metal-organic framework ratio detection fluorescent probe described in the second aspect is made into a metal-organic framework ratio detection fluorescent probe solution; the metal-organic framework ratio detection fluorescent probe solution is mixed with the water sample to be tested to obtain a mixed solution, and the fluorescence intensity of the mixed solution at 537nm and 648nm is measured at an excitation wavelength of 410nm to obtain the fluorescence intensity of Cu 2+ Fluorescence curve of concentration change, according to Cu 2+ Concentration and fluorescence ratio I 648 / I 537 The relationship between the Cu content in the water sample to be tested is calculated by 2+ concentration;

[0025] Or, the Cu 2+ Concentration and fluorescence ratio I 648 / I 537 The relationship is: y1 = 24.6069-0.8664x1, R1 2 =0.9967;y2=24.6069-0.8664x2, R2 2 =0.9994;

[0026] Among them, y1, y2 are the ratios of the fluorescence intensity at 648nm to the fluorescence intensity at 537nm, x1, x2 are Cu 2+ Concentration, where x1: 0.1-26 μM, x2: 26-33 μM;

[0027] Alternatively, the solvent of the metal-organic framework ratio detection fluorescent probe solution is deionized water, and the concentration is 40-60 μg / mL.

[0028] In a fifth aspect, the present invention provides a method for detecting copper ions in a biological sample, comprising the following steps:

[0029] The metal-organic framework ratio detection fluorescent probe described in the second aspect is made into a metal-organic framework ratio detection fluorescent probe solution; the metal-organic framework ratio detection fluorescent probe solution and the copper ion solution are added to the biological sample to be tested for incubation to obtain a mixed solution; and the fluorescent image of the mixed solution in the wavelength range of 430-600nm and 600-760nm is collected at an excitation wavelength of 410nm;

[0030] The biological sample is liver cancer cells and is not intended for disease diagnosis and treatment.

[0031] In some other embodiments, the metal organic framework ratio detection fluorescent probe solution or copper ion solution is prepared using DMEM culture medium, and the DMEM culture medium is a culture medium containing 10wt% fetal bovine serum, 1wt% penicillin and streptomycin;

[0032] The concentration of the metal organic framework ratio detection fluorescent probe solution is 40-60 μg / mL;

[0033] The concentration of the copper ion solution is 0-50 μM;

[0034] The incubation time is 1-4 hours, and after the incubation, the cells are washed with PBS solution.

[0035] Beneficial effects of the present invention:

[0036] (1) The present invention prepares the UiO-66-(OH)2@TCPP metal organic framework ratiometric fluorescent probe in one step by a solvothermal method;

[0037] (2) The present invention can not only realize the 2+ The ratiometric detection has also been successfully applied to cell imaging;

[0038] (3) The metal organic framework ratio fluorescence probe prepared by the present invention is used for Cu 2+ During detection, it has strong selectivity and anti-interference ability, high detection sensitivity and accuracy, and its detection limit is 77nM. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] The accompanying drawings in the specification, which constitute a part of the present invention, are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0040] Figure 1 Powder X-ray diffraction patterns of UiO-66-(OH)2@TCPP (a) prepared in Example 1 of the present invention, the material after UiO-66-(OH)2@TCPP was immersed in PBS buffer for 24 hours and dried (b), and the simulated UiO-66 (c);

[0041] Figure 2 Transmission electron microscopy (A) and scanning electron microscopy (B) images of UiO-66-(OH)2@TCPP prepared in Example 1 of the present invention;

[0042] Figure 3UV-visible absorption spectra of H2DHT (a), H2TCPP (b) and UiO-66-(OH)2@TCPP (c) of Example 1 of the present invention;

[0043] Figure 4 Fourier transform infrared spectra of H2DHT (a) and H2TCPP (b) and UiO-66-(OH)2@TCPP (c) of Example 1 of the present invention;

[0044] Figure 5 The UiO-66-(OH)2@TCPP and different concentrations of Cu in water in Example 2 of the present invention are shown in FIG. 2+ Fluorescence spectra of the response;

[0045] Figure 6 is the relative fluorescence intensity (I 648 / I 537 ) and Cu 2+ Linear relationship of concentration;

[0046] Figure 7 is the relative fluorescence intensity (I 648 / I 537 ) for Cu 2+ Selectivity diagram, the ordinate is the change of fluorescence intensity, and the abscissa is the number of each substance;

[0047] Figure 8 UiO-66-(OH)2@TCPP and different concentrations of Cu 2+ Response cell fluorescence imaging (a) 50μg / mL+0μM Cu 2+ ,(b)50μg / mL+20μM Cu 2+ , (c) 50 μg / mL + 50 μM Cu 2+ . DETAILED DESCRIPTION

[0048] In the embodiment of the present invention, blank (Blank), Pb 2+ , Fe 3+ 、Na + Mg 2+ , Ca 2+ , K + , Mn 2+ OH - 、F - ,I - 、NO3 - 、NO2 - , S 2- 、C2O4 2-、ClO3 - and Cu 2+ The standard solutions are obtained by dissolving PbCl2, FeCl3·6H2O, NaCl, MgCl2, Cd(NO3)2, CaCl2, KNO3, MnCl2, NaOH, NaF, NaI, NaNO2, Na2S, Na2C2O4, KClO3 and CuCl2·2H2O in water and making up the volume in a volumetric flask.

[0049] The present invention has no particular limitation on the sources of all reagents, and commercially available products known to those skilled in the art may be used.

[0050] The technical solution of the present invention is further described below in conjunction with specific embodiments.

[0051] Example 1

[0052] Preparation of UiO-66-(OH)2@TCPP

[0053] (1) Preparation of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin

[0054] Pyrrole (1.4 mL) was dissolved in 200 mL of propionic acid, and 3 g of p-formylbenzoic acid was added and dissolved under nitrogen atmosphere. The mixture was refluxed and stirred for 3 h at 150 ° C. After the reaction was cooled, it was placed in a refrigerator overnight for crystallization (0-4 ° C). Then, it was filtered and washed with dichloromethane until the color of the filtrate became lighter. Finally, it was placed under vacuum and dried at room temperature overnight to obtain 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (labeled as H2TCPP).

[0055] (2) Preparation of UiO-66-(OH)2@TCPP

[0056] At room temperature, ZrCl4 (0.164 g) and 2,5-dihydroxyterephthalic acid (0.277 g, labeled H2DHT) were dissolved in 20 mL of dimethylformamide (DMF), and then 3 mL of acetic acid and 0.2 mL of deionized water were added to form a mixed solution. After ultrasonic dispersion and complete dissolution of the solid, 90 mg of 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin (H2TCPP) was added, and the solution was stirred for 5 minutes; then, the solution was transferred to a 20 mL Teflon-lined stainless steel container and reacted in an oven at 120 °C for 24 hours. The precipitate was collected by centrifugation and washed three times with DMF. Finally, the product was completely dispersed in 50 mL of DMF for 24 hours, and UiO-66-(OH)2@TCPP was successfully obtained by centrifugation. Finally, it was placed under vacuum conditions at 60 °C and dried overnight to obtain UiO-66-(OH)2@TCPP.

[0057] A series of characterizations were carried out on the UiO-66-(OH)2@TCPP prepared in this example. Figure 1 The powder X-ray diffraction patterns of UiO-66-(OH)2@TCPP prepared in Example 1 of the present invention and the materials after UiO-66-(OH)2@TCPP was immersed in PBS buffer for 24 hours and dried, as well as the simulated UiO-66, and the powder X-ray diffraction patterns of UiO-66-(OH)2@TCPP (a) and UiO-66-(OH)2@TCPP immersed in PBS buffer for 24 hours and dried (b) and simulated UiO-66 (c). It can be seen that after UiO-66-(OH)2@TCPP was immersed in the buffer for 24 hours, its PXRD spectrum was almost unchanged, indicating that the material has good stability in the buffer.

[0058] Figure 2 Transmission electron microscopy (A) and scanning electron microscopy (B) images of UiO-66-(OH)2@TCPP; Figure 2 The SEM and TEM images show that UiO-66-(OH)2@TCPP has good dispersion. Figure 3 The UV-visible absorption spectra of H2DHT (a) and H2TCPP (b) and UiO-66-(OH)2@TCPP (c) of Example 1 of the present invention are shown in FIG. Figure 3 UV-visible absorption spectrum data showed that UiO-66-(OH)2@TCPP contained both the UV absorption characteristic peaks of H2DHT and H2TCPP, further proving the successful synthesis of UiO-66-(OH)2@TCPP.

[0059] Figure 4 Fourier transform infrared spectra of H2DHT (a) and H2TCPP (b) and UiO-66-(OH)2@TCPP (c) in Example 1 of the present invention, Figure 4 1601 and 1393cm -1 are related to the asymmetric and symmetric stretching vibration modes of the carboxyl group of the porphyrin ligand, respectively, and 1238 cm -1 The characteristic peaks are attributed to CN stretching vibration. In addition, 3100-3400cm -1 The broad and dispersed absorption peaks are caused by the stretching vibration of the associated hydroxyl groups. The stretching vibration peaks of C=O and CO of the H2DHT ligand are located at 1646 cm -1 and 1496cm -1 The above experimental results show that MOF contains dual ligands of H2DHT and H2TCPP.

[0060] Example 2

[0061] UiO-66-(OH)2@TCPP in Cu 2+ Application in detection

[0062] (1) Preparation of UiO-66-(OH)2@TCPP mother liquor

[0063] 2.5 mg of UiO-66-(OH)2@TCPP prepared in Example 1 was dispersed in 5 mL of water and dispersed evenly by ultrasonication to obtain 500 μg / mL of UiO-66-(OH)2@TCPP mother solution.

[0064] (2)Cu 2+ Detection:

[0065] 200 μL of UiO-66-(OH)2@TCPP stock solution and different volumes of CuCl2·2H2O standard solution were added to the buffer solution, and then the volume of the detection system was fixed to 2 mL (pH = 7.4) with deionized water. 2+ The final concentration of was 0-50μM, and the final concentration of UiO-66-(OH)2@TCPP was 50μg / mL. After sufficient reaction, the fluorescence emission spectrum was measured at an excitation wavelength of 410nm. The ratio of the fluorescence intensity at 648nm and the strong light intensity at 537nm was used as the ordinate, and the Cu 2+ The concentration is the horizontal axis, and the fluorescence intensity ratio -Cu 2+ Concentration relationship diagram, a linear curve was obtained by fitting.

[0066] Figure 5 UiO-66-(OH)2@TCPP and different concentrations of Cu 2+ The fluorescence spectrum of the response, as Cu 2+ As the concentration continued to increase, the fluorescence intensity at 648 nm gradually decreased, while the fluorescence intensity at 537 nm remained unchanged. Figure 6 is the fluorescence intensity ratio of UiO-66-(OH)2@TCPP (I 648 / I 537 ) and Cu 2+ The linear relationship curve of Cu 2+ Concentration and fluorescence ratio I 648 / I 537 The relationship is: y1 = 24.6069-0.8664x1, R1 2 =0.9967;y2=24.6069-0.8664x2, R2 2 =0.9994;

[0067] Among them, y1, y2 are the ratios of the fluorescence intensity at 648nm to the fluorescence intensity at 537nm, x1, x2 are Cu 2+Concentration, where x1: 0.1-26 μM, x2: 26-33 μM;

[0068] It can be seen that I 648 / I 537 With Cu 2+ There was an obvious linear relationship in the concentration ranges of 0.1-26μM and 26-33μM, and the detection limit was as low as 77nM.

[0069] Example 3

[0070] UiO-66-(OH)2@TCPP for Cu 2+ Anti-interference detection

[0071] Take several portions of 200 μL of UiO-66-(OH)2@TCPP mother solution, and then add a certain volume of Blank, Pb 2+ , Fe 3+ 、Na + Mg 2+ , Ca 2+ , K + , Mn 2+ OH - 、F - ,I - 、NO3 - 、NO2 - , S 2- 、C2O4 2- 、ClO3 - and Cu 2+ Standard solutions (numbered 1-18 in sequence, such as PO4 3- No. 1), the total volume of the detection system is 2 mL, the concentration of all interfering substances in the final system is 500 μM, Cu 2+ The concentration of 50 μM was measured at an excitation wavelength of 410 nm, and the fluorescence emission spectrum was calculated as I 648 / I 537 The fluorescence intensity ratio at the position is the basis for judging the effect of UiO-66-(OH)2@TCPP on Cu 2+ Response effect.

[0072] like Figure 7 As shown, in the Cu 2+ In the selective detection of Cu, the fluorescence intensity change at 425 nm shows that UiO-66-(OH)2@TCPP 2+ Has good selectivity.

[0073] Example 4

[0074] Cellular fluorescence imaging of copper ions by UiO-66-(OH)2@TCPP

[0075] A ratiometric fluorescence assay for Cu 2+ The method comprises the following steps:

[0076] Step S1: Cultivate liver cancer cells (HepG2) using DMEM culture medium (containing 10 wt % fetal bovine serum, 1 wt % penicillin and streptomycin).

[0077] Step S2: Prepare Cu using the above DMEM medium 2+ Solution.

[0078] Step S3: preparing a solution of ratiometric metal-organic framework fluorescent nanoprobe using the above-mentioned DMEM culture medium;

[0079] Step S4: 2+ The solution (50 μg / mL) was added to HepG2 cells, incubated in a cell culture incubator for 1 h, and washed three times with PBS (pH = 7,4);

[0080] Step S5: DMEM culture medium was then added to prepare a solution of ratiometric metal-organic framework fluorescent nanoprobes, incubated in a cell culture incubator for four hours, and washed three times with PBS (pH=7.4).

[0081] Step S6: The cells were confocal imaged under an SP8 confocal microscope, and cell fluorescence images were collected in the wavelength range of 430-600 nm (green) and 600-760 nm (red) at an excitation wavelength of 410 nm.

[0082] The concentration of the metal organic framework ratio fluorescent nanoprobe solution is 40-60 μg / mL.

[0083] Figure 8 This is the fluorescence imaging of UiO-66-(OH)2@TCPP detecting different concentrations of copper ions in HeGp2 cells. It can be seen that with the increase of copper ion concentration in the cells, the fluorescence intensity of the red channel gradually weakened, while the fluorescence intensity of the green channel remained basically unchanged, further illustrating the successful application of UiO-66-(OH)2@TCPP in cell imaging.

[0084] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A method for preparing a metal-organic framework ratio detection fluorescent probe, characterized in that: The following steps are involved: ZrCl4 and 2,5-dihydroxyterephthalic acid are dissolved in an organic solvent, acetic acid and deionized water are added to dissolve, 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin is added to dissolve, and then heated to react to prepare a metal organic framework ratio detection fluorescent probe.

2. The method for preparing a metal organic framework ratio detection fluorescent probe according to claim 1, characterized in that: The molar ratio of ZrCl4, 2,5-dihydroxyterephthalic acid and 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin is 1:2:(0.016-0.16). Or, the organic solvent is N,N-dimethylformamide; Or, the amount of acetic acid is 1-5mL; Or, the amount of deionized water used is 0-0.5 mL.

3. The method for preparing a metal organic framework ratio detection fluorescent probe according to claim 1, characterized in that: The temperature of the heating reaction is 115-125° C., and the reaction time is 22-26 hours.

4. The method for preparing a metal organic framework ratio detection fluorescent probe according to claim 1, characterized in that: After the heating reaction is completed, the obtained product is purified, and the purification process includes centrifuging, washing, soaking, washing again and drying the obtained product; The solvent used for washing or re-washing is N,N-dimethylformamide; The immersion solvent is N,N-dimethylformamide, the amount of N,N-dimethylformamide is 20-50mL, and the immersion time is 20-28h; The drying is performed at 50-70° C. in vacuum until constant weight is reached.

5. The method for preparing a metal organic framework ratio detection fluorescent probe according to claim 1, characterized in that: The preparation method of the 5,10,15,20-tetrakis(4-carboxyphenyl)porphyrin is as follows: dissolving pyrrole in propionic acid, adding p-formylbenzoic acid, and reacting at 130-160° C. for 2-4 hours under a nitrogen atmosphere to obtain the obtained product; Or, after the reaction is completed, the product may be washed and dried; The solvent used for the washing is dichloromethane, and the drying is drying at 50-70° C. under vacuum overnight.

6. A metal-organic framework ratio detection fluorescent probe prepared by the method for preparing the metal-organic framework ratio detection fluorescent probe according to any one of claims 1 to 5.

7. An application of the metal organic framework ratio detection fluorescent probe as claimed in claim 6 in the detection of copper ions in water or biological samples, when the detection sample is a biological sample, the purpose is not to diagnose and treat diseases.

8. A method for detecting copper ions in water, characterized in that: The following steps are involved: The metal-organic framework ratio detection fluorescent probe according to claim 6 is made into a metal-organic framework ratio detection fluorescent probe solution; the metal-organic framework ratio detection fluorescent probe solution is mixed with the water sample to be tested to obtain a mixed solution, and the fluorescence intensity of the mixed solution at 537nm and 648nm is measured at an excitation wavelength of 410nm to obtain the fluorescence intensity of Cu 2+ Fluorescence curve of concentration change, according to Cu 2+ Concentration and fluorescence ratio I 648 / I 537 The relationship between the Cu content in the water sample to be tested is calculated by 2+ concentration; Alternatively, the solvent of the metal-organic framework ratio detection fluorescent probe solution is deionized water, and the concentration is 40-60 μg / mL.

9. A method for detecting copper ions in a biological sample, characterized in that: The following steps are involved: The metal-organic framework ratio detection fluorescent probe according to claim 6 is made into a metal-organic framework ratio detection fluorescent probe solution; the metal-organic framework ratio detection fluorescent probe solution and the copper ion solution are added to the biological sample to be tested for incubation to obtain a mixed solution; and the fluorescent image of the mixed solution in the wavelength range of 430-600nm and 600-760nm is collected at an excitation wavelength of 410nm; The biological sample is liver cancer cells and is not intended for disease diagnosis and treatment.

10. The method for detecting copper ions in a biological sample according to claim 9, characterized in that: The metal organic framework ratio detection fluorescent probe solution or copper ion solution is prepared using DMEM culture medium, wherein the DMEM culture medium is a culture medium containing 10wt% fetal bovine serum and 1wt% penicillin and streptomycin; The concentration of the metal organic framework ratio detection fluorescent probe solution is 40-60 μg / mL; The concentration of the copper ion solution is 0-50 μM; The incubation time is 1-4 hours, and after the incubation, the cells are washed with PBS solution.

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